The detection of biological species within complex systems is important for many biomedical and environmental applications, such as infectious disease identification, medical diagnostics and therapy, and biotechnology. There is a basic interest in the development of inexpensive techniques and portable biosensors for environmental and biomedical diagnostics. Of interest is developing new techniques and sensors, not only to selectively identify target compounds, but also to assay large numbers of samples. Problems remain in reproducibly detecting and measuring low levels of biological compounds conveniently, safely and quickly.
Varieties of sensing schemes have been developed for molecular detection, such as electrochemical, optical absorption, and interferometric sensing. Fluorescence sensing remains one of the most widely used methodologies in biotechnology. Separation technologies, for example capillary array electrophoresis and micro-array technology, use fluorescent labeling for the detection of analytes such as DNA and proteins. Fluorescence detection offers single molecule sensitivity and compatibility with standard biochemical reactions, such as polymerase chain reaction (PCR).
There are several methods for selectively identifying biological species, including antibody detection and assay (e.g., Enzyme-linked Immunosuppresent Assays, or ELISA) using molecular hybridization techniques. Joining a single strand of nucleic acid with a complementary probe sequence is known as hybridization. Generally, to identify sequence-specific nucleic acid segments, sequences complementary to those segments are designed to create a specific probe for a target cell, such as a pathogen or mutant cell. Nucleic acid strands tend to pair with their complements to form double-stranded structures. Thus, a single-stranded DNA molecule (e.g., a probe), in a complex mixture of DNA containing large numbers of other nucleic acid molecules, will seek out its complement (e.g., a target). In this manner, hybridization provides an accurate way to identify very specific DNA sequences, such as gene sequences from bacteria or viral DNA. Factors impacting the hybridization or re-association of two complementary DNA strands include temperature, contact time, salt concentration, degree of mismatch between the pairs, and the length and concentration of the target and probe sequences.
The probes are typically “labeled” for easier detection of the resultant biological species after hybridization. For example, labeling the probe with a radioactive tag or marker permits subsequent detection of the radioactivity to indicate probe-target hybrids. Radioactive labeling techniques, however, suffer from several disadvantages such as limited useful lifetime for the high-energy emission isotopes used. Another method to tag probes and/or mark target compounds uses visible and/or near-infrared (NIR) dye markers for non-radioactive detection (e.g., molecular or quantum dot based probes). When hybridized to their targets, the dye-marked probes are designed to exhibit fluorescent properties when excited by certain electromagnetic radiation (e.g., laser light, lamp light, or light emitting diode (LED) light), the fluorescence being detected optically. Intensity of the fluorescence is proportional to the presence of the dye-marked probes, and can be used for quantitative measurements, and further processing. Fluorescence detection is extremely sensitive for certain target compounds; for example, a zeptomole (10−21 mole) detection limit has been realized using fluorescence detection of dyes with laser excitation. Implementation of a DNA chip based on high-density oligonucleotide arrays and fluorescence analysis is further described by Hacia et al. (J. G. Hacia, L. C. Brody, M. S. Chee. S. P. A. Fodor F. S. Collins) in Nature Genetics Dec. 14, 1996).
Despite efforts to develop chips for detection of biological species, there continues to be a need to improve implementations of micro scale detection systems for further convenience and portability. Furthermore, there continues to be a need for designs that accommodate efficient integrated circuit manufacturing techniques to realize associated cost savings.